O6-Alkylguanine-DNA alkyltransferase (AGT) is not only a key protein in protecting humans from alkylating cytotoxins and carcinogens, but it is also a predominant factor limiting the anti-tumor effectiveness of alkylating chemotherapeutic agents. In humans, AGT is a unique, direct DNA damage reversal protein that repairs O6-alkylguanine sites in a single step by selectively transferring the O6-alkyl adduct to an internal cysteine residue. This Project addresses the challenge of characterizing at the molecular level the activities of AGT relevant to its roles in genome integrity and to the knowledge-based design of AGT inhibitors and inhibitor-resistant proteins. This work will leverage and integrate the existing research strengths and programs of the investigators and their institutions to promote, develop, and test a unified understanding of AGT proteins and of novel inhibitors. The Project's four Specific Aims will 1) determine the AGT residues and motifs acting in DNA binding and conformational change, 2) establish the structural chemistry acting in damage specificity and dealkylation, 3) identify fundamental structural principles for AGT activities by in-depth comparative structural analyses of key AGT family members, and 4) create an integrated inhibitor design cycle that builds upon these coupled structural, mutational, biochemical, and computational results to develop and test new classes of AGT inhibitors. Quantitative characterization of AGT structures and protein-inhibitor complexes by X-ray diffraction, biophysical methods, and computational analyses and design in the Tainer lab will be coordinated with detailed in vitro and in vivo biochemical and mutational results from the Pegg lab. Overall, these results will provide a unified understanding of AGT relevant to characterizing its role in genetic integrity and resistance to cancer therapies. Moreover, the combination of this detailed molecular information with new inhibitors as powerful tools to knock out AGT in cells will promote knowledge-based advances directly relevant to improvements in cancer therapies.